While a great number of therapeutic compounds are discovered every year, the clinical applications of these compounds are often limited by their failure to reach the site of action. A further problem is the toxicity of many drugs at non-target sites. Often, compounds with desirable therapeutic effects have been identified and characterized only to be sidelined by their toxicity profiles. Selective drug targeting would not only reduce systemic toxicity but would also improve drug action by concentrating the therapeutic compound in selected cell or tissue targets. The delivery of drugs to specific target sites is therefore of great interest in clinical science.
Unfortunately, drug delivery technologies have not kept pace with target identification and novel compound synthesis. Delivery problems are especially lacking in the rapidly evolving area of RNA and DNA-based therapeutic intervention.
Increasingly, liposomes are being used to deliver drugs and other agents to target sites in cells. Liposomes are hollow, spherical vesicles comprised of membranes that behave as two-dimensional fluids. In a spherical model, steric stability is heightened at a particular particle diameter for any particular lipid formulation based on the free energies associated with slight deformations of the membrane. Adsorption, spreading, fusion, self-healing, and other mechanical properties of liposomes are recognized as important performance indicators toward their application as delivery vehicles.
In general, liposomes can be formed with outer diameters ranging from 20 to 1000 nm (1 μm), more typically 40-500 nm, with ˜100 nm diameter liposomes being particularly desirable for many biological applications. Liposomes smaller than about 20 nm are physically untenable, while liposomes larger than about 1 μm in diameter tend to be unstable and aggregate over time. Liposome size has a direct effect on payload encapsulation efficiency in the case of an active loading scheme whereby preformed liposomes absorb active ingredients from the surrounding media into their interiors, with smaller sized vesicles being more efficient than larger ones. To a large extent, liposome size determines the sites of action of liposome-cell interaction. Size affects not only how and where the liposomes enter a cell, but also whether they reach a particular cell at all. For some therapeutic applications, efficient tissue targeting requires that the liposomes be able to circulate in the bloodstream for a long period of time until a proper target is encountered.
In vivo, liposomes that are too large as a direct result of the manufacturing process, or that agglomerate into larger units as a result of secondary instabilities in solution, will tend to become entrapped in areas simply based on size. For example, the liver removes larger particles from the bloodstream (larger than 200 nm diameter) because of vasculature sized to act as a physical filter. For this reason, many liposome formulations have been created with liver tissue targeting in mind simply because large particles end up in the liver, and this observation leads to the illusion of a natural affinity of liposomes for liver cells. In fact, oversized liposomes merely become entrapped in the liver because of their size. In any application not targeting the liver, liver localization would have the detrimental effect of removing active material from the intended site of deposition, as well as increasing the likelihood of off-targeting and side effects by misplacing an otherwise therapeutic payload.
In some therapeutic applications, liposomes are administered by intravenous injection, and liposome size—and charge—directly influence the clearance of liposomes from the patient's bloodstream. Generally, the longest half-lives are obtained when liposomes are small in diameter (<0.05 μm). It has also been found that “liquid” vesicles are more rapidly removed from blood circulation than “rigid” ones. The behavior of liposome preparations given by alternative parental routes, such as intraperitoneal, subcutaneous or intramuscular route is also influenced by the distribution of liposome size.
In many therapeutic applications, and particularly in systemic delivery and tissue and cell targeting, liposome size is a critical parameter of therapeutic effectiveness. In order for liposomes to function efficiently as vectors for a given biological application, they need to be as monodisperse as possible, i.e., have as narrow a size distribution as possible. In general liposomes are measured in terms of their (outer) diameters, with little discussion in the literature of internal volume. The literature suggests that a collection of liposomes is considered uniformly sized if the liposomes' outer diameters are polydisperse by only ±10%, i.e., 90-110 nm outer diameters for a collection of liposomes having a mean diameter of 100 nm. The fact that this is considered “good” is shocking, as a difference of 10% in diameter corresponds to roughly a 92% difference in internal volume (if, e.g., one assumes an 8 nm thick lipid layer).
Obtaining the ideal liposome size is therefore a matter of determining the proper chemistry for a given biological application and sizing the particles at exactly those dimensions—a tall order for existing technologies.
Clearly, liposome size distribution is a critical parameter with respect to the pharmacological and pharmacodynamic behavior of biologically active substances that are site-specific targeted in vivo. Although various methods of making small unilamellar vesicles (SUVs) are available, from a process perspective, the formation of stable SUVs with a narrow and predictable size distribution remains a challenge. Commercial liposome sizing systems typically operate by making a number of passes through various size reduction methodologies, that utilize shear force and/or ultrasonic energy dispersion to reduce the size of the liposomes to an approximated average. The most common means of resizing is by passing the liposomes a number of times through a membrane. The production of liposomes with very true homodispersity (i.e., substantially monodisperse), has not been reported, and there is no protocol available in the literature for the production of such particles, let alone a protocol for achieving narrow size distributions under the demanding conditions and in the large volumes required for pharmaceutical production.
An unexpected benefit of the regular sizing of liposomes is the ability to control charge density. Charge density is determined by both the internal payload and external lipid envelope. The lipids comprising the envelope are chosen according to their charge, and the ratio of the constituent lipids is determined according to the charge desired. Determining and quantizing the desired overall charge of the loaded particle is particularly important for delivery of highly charged payload such as DNA. Since DNA payloads are often large, and a single copy of the DNA is loaded per liposome, the negative charge is best neutralized by an envelope of a specific size in order to achieve a desired charge balance. Slight variations in charged liposome size distribution could therefore profoundly affect biodistribution. Considering this fact, and not anticipating that liposomes could be made to have a very uniform size/charge, one author wrote that this factor will serve to “preclude or at least limit the in vivo use of many potentially effective lipid-based DNA delivery vectors.”
The limitations of current technology have a detrimental impact on clinical research and commercial utilization of liposome treatments. When polydisperse liposome formulations are used, valuable markers, isotopes, drugs, and other reagents and payloads are wasted, as they do not reach their intended target and are effectively lost. This retards the development of new therapies (in terms of wasted opportunities and increased time in the lab), and increases the cost of commercial applications (more liposomes are required, as much of the liposomes are the wrong size to be effective).
Accordingly, there is a very strong need in the pharmaceutical, biotechnology, and cosmetics industries for substantially homogenous liposome formulations, particularly unilamellar liposomes that exhibit diameters in the 100 to 200 nm range, and an efficient, robust system for reproducibly generating uniformly sized liposomes and other small particles. In addition, with current liposome and particle manufacturing techniques, it is exceedingly difficult, if not impossible, to know exactly—or even approximately—how many particles are in a given container of any size. This is because available manufacturing processes are batch processes, and only after the batch is created can a person find out what the yield was, and this is accomplished by running a sample through a particle size analyzer (PSA), or by doing some electron microscopy. Both of these methods are expensive, error-prone, and generally unreliable. A digital manufacturing process would be a significant improvement over the art, as it would enable liposomes and other small particles to be produced with great accuracy and precision.